Use of sodium citrate anticoagulant for routine hematology - Carden ...

ISLHLaboratory Hematology 4:156–162© 1998 Carden Jennings Publishing Co., Ltd.#98-1.Perrotta.Spring98Official PublicationUse of sodium citrate anticoagulant for routinehematology analysis on the CELL-DYN ® 4000:An opportunity to enhance efficiency in theclinical laboratoryG. PERROTTA, 1 L. ROBERTS, 1 J. GLAZIER, 2 H.R. SCHUMACHER 11 Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH2 Abbott Laboratories, Abbott Park, ILABSTRACTBlood from 67 sophomore medical students was used to evaluatedifferences in complete blood count (CBC) normal ranges inspecimens collected in either liquid tripotassium ethylene diaminetetraacetic acid (EDTA) or liquid trisodium citrate and analyzedby the Abbott CELL-DYN 4000 hematology system (AbbottDiagnostics, Abbott Park, IL). The data support the premise thatcitrate is a suitable anticoagulant for routine CBC analysis providedthat a dilution correction factor for counts, hematocrit, andhemoglobin is applied automatically, either as a built-in algorithmin the software of the cell counter or of the laboratory informationsystem. Once the corrections were made, the results showed excellentagreement between the two anticoagulants. In addition,peripheral blood smears prepared from citrated blood showedgood morphology. Overall, the data demonstrate the suitability ofperforming a CBC with a citrated specimen, once it has been correctedfor the dilution. This observation could facilitate the adaptationof one tube for two major analyses (hematology and coagulationparameters), especially considering the advances in informaticsand robotics technology. Immediate operational efficienciescould be realized throughout the testing process. Some would generatecost savings by eliminating the EDTA tube for routine CBCtesting. Other savings could be generated in the pre-analyticalphase, principally in the specimen collection process because fewertubes and labels, and decreased sorting, routing, and phlebotomytime would be required. The authors suggest that larger and morethorough studies be performed to ascertain that these findings inAddress correspondence to Gerardo Perrotta, MPA, Department of Pathologyand Laboratory Medicine, University of Cincinnati, PO Box 670529, Cincinnati,OH 45267-0529.Received 29 January 1998; accepted 3 April 1998citrated samples are valid across a broad spectrum of hematologyconditions, and that they can be easily generated in the evolvingand reconfigured laboratory operations. Lab Hematol 4:156–162, 1998KEY WORDS: Automated CBC · EDTA ·Hematology analyzersINTRODUCTIONThe purpose of this study was to compare tripotassium ethylenediamine tetraacetic acid (K 3 EDTA) with trisodium citrate collectiontubes in regard to automated hematology parameters, and toevaluate the latter for reliablity and effectiveness. Using a single tubefor two major tests in the clinical laboratory promotes both operationaland technical efficiencies. Advances in informatic and robotictechnology present opportunities or reasons to evaluate other anticoagulantsthat have been considered impractical or expensive [1,2].The use of trisodium citrate requires correcting the dilution eithermanually or by transfer to another personal computer and wouldsubstantially affect the turnaround time of coagulation testing givenpresent-day laboratory configurations. Heparin has always beenconsidered expensive and to interfere with the staining properties ofcells. The oxalates cause cell shrinkage or swelling, depending onthe potassium or ammonium salt used.Thus, given its general availability, ease of preparation, widespreaduse, and relatively low cost, a salt of EDTA has become theanticoagulant of choice. The National Committee for Clinical LaboratoryStandards (NCCLS) H1-A4 standard [3] recommendsEDTA as the preferred anticoagulant for complete blood countanalysis and WBC differential, principally for its overall cell preservationproperties. However, an attempt to select and standardize thedry powder (di-sodium or dipotassium) or liquid (tripotassium) saltof EDTA through a tentative standard (H35-T) process has beendiscontinued and replaced by the H1-A standard. For its part, the156

Sodium Citrate CBCs on the CD-4000 157International Council for Standards in Hematology (ICSH) ExpertPanel on Cytometry has evaluated the three EDTA salts and recommendsK 2 EDTA [4]. In the United States, 50% of laboratoriesuse the recommended anticoagulant. Regardless of its current status,many practitioners appreciate certain limitations of EDTA, whichstem from a wide range of additives (powder, liquid, spray; di- ortri-salts of sodium or potassium) and evacuated tube sizes and typesstill available in the market. These variables coupled with inconsistentspecimen volume draws and a variety of mixing, storing, andtransporting conditions produce questionable results [5–7].On the other hand, sodium citrate would preclude many of thevariables mentioned above. Because sodium citrate has been used asan anticoagulant primarily for coagulation studies, phlebotomistsare attuned to the full draw requirement of the coagulation (bluetop)tubes regardless of their size. The ratio of anticoagulant toblood is better maintained with citrate than with EDTA, thusachieving a greater standardization currently lacking with EDTA.The growing complexity of modern laboratory systems and thegreater emphasis on developing cost-efficient and innovative solutionsto numerous problems increase the need to explore and developnew concepts without sacrificing quality across the spectrum ofcare. At our institution, approximately 150,000 CBCs and 50,000coagulation tests (prothrombin time [PT] and activated partialthromboplastin time [aPTT]) are performed annually. Up to 25%of the CBCs are accompanied by a coagulation request; conversely,80–90% of the routine coagulation tests are accompanied by aCBC request. If these CBC and coagulation requests were submittedin a single tube, significant efficiencies and cost benefits couldbe realized by an increased use of an expanding biotechnology andinformatics industry in the clinical laboratory.MATERIALS AND METHODSBlood SamplesPeripheral blood samples from 67 second-year medical students(42 men, 25 women), ranging in age from 19 to 41 years (mean of25 years) were used for this study.Specimens were collected in 3-mL vacutainer Hemogard tubes(K 3 EDTA; Becton Dickinson, Franklin Lakes, NJ) and 5-mLblue-top tubes (3.2% trisodium citrate; Becton Dickinson) using21-gauge 1.5" needles. Specimens were mixed by gentle inversion,labeled, and prepared for analysis. Only paired specimens (i.e., oneK 3 EDTA and one trisodium citrate sample from the same student)were used for analysis. Samples that did not meet minimum volumerequirements were discarded. All tubes were bar coded andplaced on the autosampler for automatic processing.CBC profiles were generated, first for the citrated and then theEDTA tubes. All specimens were kept at room temperaturethroughout the study. Results were completed within 7 hours ofcollection and stored in the data management system of the CELL-DYN 4000 (Abbott Diagnostics, Santa Clara, CA). Peripheralblood smears were prepared within 3 hours of collection andstained with Wright’s stain in an Ames Hematek stainer (Miles,Diagnostic Division, Elkhart, IN).Hematology AnalyzerUsing the software package R7-3 and reagents supplied by AbbottDiagnostics, the CELL-DYN 4000 was calibrated according to manufacturerguidelines and produced results within the acceptable rangeon all control samples. Three levels of controls (low, normal, andhigh) were within established limits for that specific lot number. Parallelquality control assessment with the CELL-DYN 3500 revealedthat the two instruments were in agreement for all values.Suspect flags were preset by the manufacturer and the high-lowdistributional flags were defined and preset by the laboratory.The CELL-DYN 4000 is the latest fully automated hematologyanalyzer that uses four-angle argon-laser light scatter, two-color fluorescence,high-resolution focused flow impedance, and hemoglobinspectrophotometry [8].Peripheral Blood SmearsPush wedge slides were prepared manually and stained withWright’s-Giemsa on the Hematek stainer (Miles). The slide preparedfrom the EDTA sample was used as the control. All cell lineswere examined for the EDTA and citrate samples using magnification20 and oil immersion with constant light intensity. A blindedstudy was conducted to determine if one (H.R.S.) could identifywhether a slide was from EDTA or citrate origin.Data ManagementData were generated by and stored in the data management systemof the CELL-DYN 4000. It was later downloaded on diskettefrom the CELL-DYN 4000 Datalog in accordance with a protocolprovided by Abbott [9]. Corrections for cell counts and hemoglobinmeasurements were performed with Microsoft Excel 5.0(Microsoft, Redmond, WA).The WBC, red blood cell count (RBC), hemoglobin (Hb),hematocrit (HCT), and platelet (Plt) results were multiplied by afactor of 1.1 to correct for sample dilution. The absolute leukocytecounts (neutrophils, lymphs, monocytes, eosinophils, andbasophils) also were multiplied by the same factor; however, thepercent leukocytes were recalculated by a simple percent ratio ofabsolute leukocyte to total WBC 100.Sized parameters (mean corpuscular volume [MCV], red bloodcell distribution width [RDW], and mean platelet volume [MPV])and proportional parameters (mean corpuscular hemoglobin[MCH] and mean corpuscular hemoglobin concentration[MCHC]) required no correction because these measurements wereunaffected by dilution.StatisticsThe results produced from analyses and corrected parameters wereanalyzed by descriptive statistics to determine standard deviations andmeans for each parameter. Multiple regression analysis (R 2 ) was usedto compare counts, paired Student’s t-test to compare means andRDW, and chi-square to evaluate peripheral blood smears.Statistical analyses and evaluations were calculated using theMicrosoft Excel 5.0 and Jandel SigmaStat 2.0 (SPSS, Chicago, IL).ResultsData obtained in the present study with the resulting paired dataanalyses (R 2 for counts and p for means and distribution widths) arelisted in Table 1. Results from the citrated samples are corrected forthe dilution. The mean and range of all parameters determinedfrom the citrated samples compare well with those from the EDTAsamples, except for the parameters marked with an asterisk. Thesehighlighted parameters show a poor statistical correlation for theabsolute basophil counts of females and monocyte counts of the

158 G Perrotta et al.TABLE 1. CBC profile parameters and comparative ranges using the CELL-DYN ® 4000; EDTA vs. citrateWomen (n=25)Men (n=42)Mean ± 2SD rangeMean ± 2SD rangeAnalyte SI unit EDTA Citrate ( 1.1) R 2 EDTA Citrate ( 1.1) R 2WBC 10 9 /L 5.9 (2.9–8.9) 6.0 (2.9–9.1) 0.88 5.8 (3.4–8.3) 5.6 (3.3–8.0) 0.94Neutrophil 10 9 /L 3.4 (1–5.8) 3.5 (1–6) 0.91 3.2 (1–5.5) 3.1 (0.9–5.3) 0.98Lymphocyte 10 9 /L 1.9 (1–2.8) 2 (1–2.9) 0.89 1.9 (1.2–2.6) 1.9 (1.2–2.6) 0.84Monocyte 10 9 /L 0.4 (0.1–0.7) 0.4 (0.2–0.7) 0.83 0.5 (0.3–0.8) 0.4 (0.1–0.7) 0.54 aEosinophil 10 9 /L 0.1 (0–0.3) 0.1 (0–0.3) 0.84 0.2 (0–0.4) 0.2 (0–0.3) 0.86Basophil 10 9 /L 0.03 (0–0.08) 0.03 (0–0.07) 0.00 a 0.04 (0–0.1) 0.04 (0–0.12) 0.86RBC 10 12 /L 4.52 (3.76–5.28) 4.5 (3.66–5.35) 0.93 4.99 (4.0–5.98) 5.04 (4.09–5.98) 0.90Hb g/dL 13.8 (12–15.5) 13.8 (11.6–16) 0.95 15.3 (12.3–18.2) 15.6 (12.9–18.2) 0.80HCT % 40.3 (35–45.5) 40.8 (34.9–46.7) 0.87 44.5 (36.4–52.6) 45.2 (37.8–52.6) 0.89Plt 10 9 /L 240 (150–329) 231 (139–323) 0.89 222 (134–311) 210 (136–285) 0.96p ≤ 0.05 p ≤ 0.05Neutrophil % 57.3 (40.1–73.9) 57 (39.7–74.3) 0.284 54.1 (37.3–70.9) 54.5 (38–71.1) 0.082Lymphocyte % 32.7 (18.2–47.2) 33.3 (18–48.6) 0.912 33 (18.6–47.4) 34.7 (19.6–49.7)

160 G Perrotta et al.Cell(s) EDTA CitrateNeutrophil/Platelet/RBCABEosinophilCDBasophilEFLymphocyteGHMonocyteIJFIGURE 1. Characteristic view of cells in EDTA vs. citrate oil immersion (1000).samples, however, was considered impractical [1]. Recently, Gulatiet al. [13] suggested mixing the blood by vortex to disaggregateplatelet clumps. Lippi et al. [12] have formulated a dry citrate-pyridoxal5 phosphate (CPT) anticoagulant to improve cell countsand sizing, especially for platelets and MPV. Although these studiesfocus on improving platelet counts, we maintain that adopting theexisting sodium citrate tube for CBC analysis holds greater benefitfor routine laboratories as they continue to undergo major reengineeringto achieve the highest efficiencies at the lowest operationalcost. In our view, CPT anticoagulated samples could not be easilyadopted for routine coagulation testing within the same time framerequired for the trisodium citrate samples. All reference ranges for

Sodium Citrate CBCs on the CD-4000 161EDTACitrateABFIGURE 2. Characteristic scatterplots and histograms of EDTA vs. citrate.coagulation tests would have to be determined on CPT samples—amore costly and laborious proposition with far-reaching impact onpatient care (i.e., monitoring Coumadin and heparin therapies).To our knowledge, no other studies have approached the questionof performing CBCs from sodium citrate tubes to improvework flow and cost containment and to facilitate further laboratoryreengineering to accommodate the expanding role of robotics andinformation technology.Medical students represent a normal group for establishing initialcomparability and acceptability and determining the feasibilityof adopting an unconventional anticoagulant for CBC parameters.Because medical students represent a distinct and healthy agegroup,however, similar studies with other age-groups and varyingclinical conditions and pathologies will be required.The evolution of the hematology analyzer is converging withthat of informatics and robotics. Expansion of bio-informatics technologymakes it possible to alter past methods for more practicalapplications in today’s cost-restrictive health care environment [14].This study promotes the idea that CBCs can be performed oncitrated samples provided that manufacturers of hematology analyzersincorporate algorithms or software programs that automaticallycorrect for the citrate dilution. These programs can be located withineither the instrument data management system or the laboratoryinformation system. All parameters reported per unit volumewould require a multiplication factor of 1.1 to account for the dilutionof the sample. Mean and proportional parameters can bedirectly reported. Additional benefits also may be realized forpatients and laboratories.The amount of blood needed for the most frequently orderedand fully automated test would be decreased. This reduced amountof blood translates into good public relations because of patient concernsabout the amount of blood drawn per test, and offers betterpatient care and reduced risk of producing iatrogenic anemia. Operationally,the pre-analytic phase of a CBC will undergo a reductionin collection time. For example, less time will be spent on the phlebotomyprocedure, thus freeing the phlebotomists or nurse assistantsto perform other ward responsibilities. Consequently, it may reducethe number of phlebotomists needed, thus generating more savings.Within the laboratory, the pre-analytic phase also would be curtailedbecause there would be fewer tubes to handle and process,favoring a more mechanized operation. This would result in technicaltime savings. Other cost factors also would be significantly and favorablyaffected. Inventory cost for lavender-top tubes and labels wouldbe reduced by the number of CBCs performed. For example, in ourinstitution the conversion to a single citrate tube would generate asavings of $2000 per annum based on 150,000 CBCs and 50,000routine coagulation tests performed annually at a cost of $0.06 and$0.07 for each tube needed for the tests, respectively. Other savingsmay be generated by a reduction of infectious waste that many laboratoriespay to have hauled away. In our area, the cost of infectiouswaste disposal ranges from $0.10 to $0.30 per pound, depending onwhether it is pre-treated (sterilized on site) before discarding.More importantly, performing CBCs and coagulation testingfrom one tube positions the laboratory to improve efficiency andthus become more cost-effective when it transitions to a fully automated,roboticized, and informatics-driven system. Several vendors

162 G Perrotta et al.TABLE 4. LimitationsSample questions to be addressed in future studies1. Does the observation made on the CELL-DYN 4000 hold true forother hematology instruments?2. Is the sample stability of WBC, RBC, and Plt parameters similar incitrate and EDTA in terms of time intervals (0–24 or 72 hours),temperature (room vs. refrigerator), transportation (manual vs.automated), and quantity or volume of sample?3. What is the impact on coagulation testing after a sample has beenprocessedfor CBC analysis, e.g., mixing and cap piercing?4. What protocols should be established for rechecking CBC orcoagulation results?5. What laboratory re-design would be needed to facilitate workflowprocesses for aliquoting, sorting, spinning and sampling?6. What would be the most efficient algorithm to adopt for correctingthe dilution effect of citrate, i.e., within the data managementsystem of the analyzer or of the LIS?7. What changes are needed in bar code systems to print labels withcodes for EDTA or citrate, for combined CBC and coagulant testingwith or without differential, CBC, or hemoglobin, and forhematocrit only and coagulation (PT, APTT, or fibrinogen) only?are introducing consolidated hematology workstations that areoperated by robotics and information technology. Although not yetavailable, one could add a coagulation analyzer to this workstationand have the CBCs and coagulation tests performed from the samevial with the aid of computerized (programmed) articulated robots.Technically, the use of citrated samples provides opportunites toexpand and improve evaluation of platelets. It also would open newavenues to investigate whether sodium citrate is acceptable for thepotential incorporation of limited cytometric functions(immunophenotyping) with the more advanced hematology analyzerssuch as the CELL-DYN 4000.The data presented support the premise that sodium citratesamples provide similar interpretation of results within the normalrange when compared with previously established ranges. Becausethis conversion would happen over time, work flow systems wouldbe designed to accommodate both anticoagulants for a while. Thesesystems may be achieved with existing bar code technology, whichcurrently deciphers whether a CBC gets a differential.The observation made in this study needs to be confirmedacross the wide range of hematology conditions and varying laboratorypractices and instrumentations.Additional and more comprehensive studies are necessary toaddress the limitations of this study, as listed in Table 4.REFERENCES1 KOEPKE JA: Tips on technology. MLO Med Lab Obs 13:15, 19812 CARTWRIGHT GE: Diagnostic Laboratory Hematology, 4th edition. NewYork, NY, Grune & Stratton, 1968, p. 273 NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS: Evacuatedtubes and additives for blood specimen collection, 4th edition.Approved Standard. NCCLS Document H1-A4, Villanova, PA, 19964 INTERNATIONAL COUNCIL FOR STANDARDIZATION IN HEMATOLOGY:Expert panel on cytometry recommendations of the International Councilfor Standardization in Hematology for ethylene diamine tetraacetic acidanticoagulation of blood for blood cell counting and sizing. Am J ClinPathol 100:371, 19935 SAVAGE RA: Pseudoleukocytosis due to EDTA-induced platelet clumping.Am J Clin Pathol 81:317, 19846 RABINOVITCH A: Anticoagulants, platelets and instrument problems. Am JClin Pathol 82:132, 19847 KOEPKE JA: Laboratory Hematology. New York, NY, Churchill Livingstone,19848 CELL-DYN ® 4000: Principles of Operation, revision 3. Abbott Park, IL,Abbott Diagnostics, 19979 CELL-DYN ® 4000: DataLog Extract Procedure for the Efficiency Evaluationof the CELL-DYN ® 4000. Abbott Park, IL, Abbott Diagnostics, 199710 HUYN BH, ASHTON JK, DOLAN K: Practical Hematology: A LaboratoryGuide With Accompanying Filmstrips. Philadelphia, PA, WB Saunders, 197511 BRUNSON D, SMITH D, BAK A, PRZYK E, SHERIDAN B, MUNCER D: Comparinghematology anticoagulants: K2EDTA vs. K3EDTA. Lab Hematol1:112, 199512 LIPPI U, SCHINELLA M, MODENA N, NICOLI M, LIPPI G: Advantages of anew anticoagulant in routine hematology on the Coulter Counter S PlusSTKR analyzer. Am J Clin Pathol 93:760, 199013 GULATI GL, ASSELTA A, CHEN C: Using vortex to disaggregate plateletclumps. Lab Med 28:665, 199714 WILKINSON DS: The role of technology in the clinical laboratory of thefuture. Clin Lab Manage Rev 11:322, 1997